Bioinformatics and Expression Pattern Analysis of HvnJAZ4 Gene in Hulless Barley

doi:10.13560/j.cnki.biotech.bull.1985.2024-0479

Abstract

Abstract:

【Objective】The JAZs protein family is an important component of the jasmonic acid signaling pathway in plants. This study aimed to investigate the gene and protein structure and molecular dynamics of HvnJAZ4. Exploring the HvnJAZ4 gene and protein structure, molecular dynamics and expression pattern characteristics of JA signal inhibitor in hulless barley may provide a reference for the study of the regulatory mechanism of the hulless barley HvnJAZs gene family.【Method】Different bioinformatics methods were used to analyze the gene and protein structure of HvnJAZ4 as well as molecular dynamic characteristics. qPCR and subcellular localization techniques were used to study their expression pattern.【Result】The HvnJAZ4 promoter region contained cis-acting elements related to the responses to jasmonic acid（JA）, abscisic acid（ABA）, gibberellin（GA）, and salicylic acid（SA）, and low temperature. Further, the HvnJAZ4 protein contained 418 amino acids, with α-helix, extended strand, β-sheet, and random coil by the proportions 23.50%, 12.47%, 2.64%, and 61.39%, respectively. HvnJAZ4 showed the closest homology to the TdJAZ4 protein in Triticum dicoccoides. HvnJAZ4 contained typical NT, ZIM, and Jas domains, while the ZIM domain is the key domain for HvnJAZ4 to interact with other HvnJAZs and HvnNINJA1, and the Jas domain is the key domain for HvnJAZ4 to interact with HvnCOI1b. Molecular dynamics simulations identified that one key amino acid site interacting with JA-Ile, and eight key sites of HvnJAZ4·Jas interacting with JA-Ile, and eight key sites interacting with HvnCOI1b. Compared to the leaves, roots, stems and stem internodes, HvnJAZ4 was highly expressed in grains and tiller buds, and its expression was induced by low temperature, MeJA, ABA, GA. Subcellular localization results indicated that HvnJAZ4 was localized in the nucleus.【Conclusion】HvnJAZ4 in hulless barley may play a key role in regulating growth, development, and stress response through JA signaling and synergistic interactions with other plant hormones.

Key words: hulless barely, HvnJAZ4 protein, bioinformatics analysis, molecular dynamics simulation

WANG Zi-ao, TIAN Rui, CUI Yong-mei, BAI Yi-xiong, YAO Xiao-hua, AN Li-kun, WU Kun-lun. Bioinformatics and Expression Pattern Analysis of HvnJAZ4 Gene in Hulless Barley[J]. Biotechnology Bulletin, 2025, 41(1): 173-185.

Figures/Tables 11

References 36

[1]	安立昆, 姚有华, 姚晓华, 等. 青稞耐低氮相关类甜蛋白基因HvTOND1克隆和亚细胞定位研究[J]. 西北农业学报, 2021, 30(8): 1157-1166.
	An LK, Yao YH, Yao XH, et al. Cloning and subcellular localization of related thaumatin-like protein gene HvTOND1 tolerant to low nitrogen in hulless barley[J]. Acta Agric Boreali Occidentalis Sin, 2021, 30(8): 1157-1166.
[2]	任晴雯, 安立昆, 姚有华, 等. 青稞HvnPHO1;2基因克隆、亚细胞定位和表达模式分析[J]. 西北农业学报, 2021, 30(10): 1461-1472.
	Ren QW, An LK, Yao YH, et al. Cloning,subcellular localization and expression analysis of phosphate transporter gene HvPHO1;2 in hulless barely[J]. Acta Agriculturae Boreali - occidentalis Sinica, 2021, 30(10): 1461-1472.
[3]	颜昌兰. 青稞品种稳定性、适应性及主要农艺性状的评价与分析[D]. 杨凌: 西北农林科技大学, 2015.
	Yan CL. Evaluation and analysis of stability, adaptability and main agronomic characters of highland barley varieties[D]. Yangling: Northwest A&F University, 2015.
[4]	弓开元. 青藏高原青稞产量和光温生产潜力对气候变化的响应[D]. 杨凌: 西北农林科技大学, 2020.
	Gong KY. Response of highland barley yield and light and temperature production potential to climate change in Qinghai-Tibet Plateau[D]. Yangling: Northwest A&F University, 2020.
[5]	张银乐, 张文静, 杨洋, 等. 低温对青稞种子萌发及幼苗生长的影响[J]. 安徽农学通报, 2018, 24(16): 28-31.
	Zhang YL, Zhang WJ, Yang Y, et al. Effects of low temperature on seed germination and seedling growth of highland barley[J]. Anhui Agric Sci Bull, 2018, 24(16): 28-31.
[6]	王玉林, 徐齐君, 原红军, 等. 西藏青稞品种喜马拉雅8号低温处理的SSH-cDNA文库构建及分析[J]. 麦类作物学报, 2017, 37(8): 1025-1030.
	Wang YL, Xu QJ, Yuan HJ, et al. Construction and analyses of SSH-cDNA library of Tibetan hulless barley(Hordeum vulgare l. var. nudum HK.f.)ximalaya 8 under low temperature treatment[J]. J Triticeae Crops, 2017, 37(8): 1025-1030.
[7]	Yan YX, Christensen S, Isakeit T, et al. Disruption of OPR7 and OPR8 reveals the versatile functions of jasmonic acid in maize development and defense[J]. Plant Cell, 2012, 24(4): 1420-1436.
[8]	An LK, Ahmad RM, Ren H, et al. Jasmonate signal receptor gene family ZmCOIs restore male fertility and defense response of Arabidopsis mutant coi1-1[J]. Plant Growth Regul, 2019, 38(2): 479-493.
[9]	Chini A, Fonseca S, Fernández G, et al. The JAZ family of repressors is the missing link in jasmonate signalling[J]. Nature, 2007, 448(7154): 666-671.
[10]	Chung HS, Cooke TF, Depew CL, et al. Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling[J]. Plant J, 2010, 63(4): 613-622.
[11]	孙程, 周晓今, 陈茹梅, 等. 植物JAZ蛋白的功能概述[J]. 生物技术通报, 2014(6): 1-8.
	Sun C, Zhou XJ, Chen RM, et al. Comprehensive overview of JAZ proteins in plants[J]. Biotechnol Bull, 2014(6): 1-8.
[12]	Thines B, Katsir L, Melotto M, et al. JAZ repressor proteins are targets of the SCF^COI1 complex during jasmonate signalling[J]. Nature, 2007, 448: 661-665.
[13]	Oblessuc PR, Obulareddy N, DeMott L, et al. JAZ4 is involved in plant defense, growth, and development in Arabidopsis[J]. Plant J, 2020, 101(2): 371-383. doi: 10.1111/tpj.14548
[14]	晏胜伟, 孙程, 周晓今, 等. 玉米JAZ家族基因ZmJAZ4的克隆及功能分析[J]. 生物技术通报, 2015(3): 96-101. doi: 10.13560/j.cnki.biotech.bull.1985.2015.04.013
	Yan SW, Sun C, Zhou XJ, et al. Cloning and characterization analysis of ZmJAZ4, a JAZ family gene in maize[J]. Biotechnol Bull, 2015,(3): 96-101.
[15]	王世通. 基于PhJAZ4转基因的矮牵牛新种质创制与评价[D]. 武汉: 华中农业大学, 2023.
	Wang ST. Creation and evaluation of new petunia germplasm based on PhJAZ4 transgenic[D]. Wuhan: Huazhong Agricultural University, 2023.
[16]	杨娆. 丹参SmJAZ4调控酚酸类物质合成的分子机制[D]. 西安: 陕西师范大学, 2022.
	Yang R. Molecular mechanism of the regulation of phenolic acid synthesis by Salvia miltiorrhiza SmJAZ4[D]. Xi'an: Shaanxi Normal University, 2022.
[17]	Miccono MLA, Yang HW, DeMott L, et al. Review: Losing JAZ4 for growth and defense[J]. Plant Sci, 2023, 335: 111816.
[18]	Zhang MF, Luo X, He W, et al. OsJAZ4 fine-tunes rice blast resistance and yield traits[J]. Plants, 2024, 13(3): 348.
[19]	DeMott L, Oblessuc PR, Pierce A, et al. Spatiotemporal regulation of JAZ4 expression and splicing contribute to ethylene- and auxin-mediated responses in Arabidopsis roots[J]. Plant J, 2021, 108(5): 1266-1282.
[20]	Sheard LB, Tan X, Mao HB, et al. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor[J]. Nature, 2010, 468(7322): 400-405.
[21]	Abramson J, Adler J, Dunger J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3[J]. Nature, 2024, 630(8016): 493-500.
[22]	Sanner MF. Python: a programming language for software integration and development[J]. J Mol Graph Model, 1999, 17(1): 57-61. pmid: 10660911
[23]	Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility[J]. J Comput Chem, 2009, 30(16): 2785-2791. doi: 10.1002/jcc.21256 pmid: 19399780
[24]	Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[J]. J Comput Chem, 2010, 31(2): 455-461. doi: 10.1002/jcc.21334 pmid: 19499576
[25]	Pierce LCT, Salomon-Ferrer R, Augusto F de Oliveira C, et al. Routine access to millisecond time scale events with accelerated molecular dynamics[J]. J Chem Theory Comput, 2012, 8(9): 2997-3002. pmid: 22984356
[26]	Götz AW, Williamson MJ, Xu D, et al. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. generalized born[J]. J Chem Theory Comput, 2012, 8(5): 1542-1555. pmid: 22582031
[27]	Salomon-Ferrer R, Götz AW, Poole D, et al. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. explicit solvent particle mesh Ewald[J]. J Chem Theory Comput, 2013, 9(9): 3878-3888. doi: 10.1021/ct400314y pmid: 26592383
[28]	Wang JM, Wolf RM, Caldwell JW, et al. Development and testing of a general amber force field[J]. J Comput Chem, 2004, 25(9): 1157-1174. doi: 10.1002/jcc.20035 pmid: 15116359
[29]	Wang JM, Wang W, Kollman PA, et al. Automatic atom type and bond type perception in molecular mechanical calculations[J]. J Mol Graph Model, 2006, 25(2): 247-260. pmid: 16458552
[30]	Sousa da Silva AW, Vranken WF. ACPYPE-AnteChamber PYthon parser interface[J]. BMC Res Notes, 2012, 5: 367. doi: 10.1186/1756-0500-5-367 pmid: 22824207
[31]	魏昕, 刘雨恒, 刘宇阳, 等. 植物JAZ蛋白家族研究进展[J]. 植物生理学报, 2021, 57(5): 1039-1046.
	Wei X, Liu YH, Liu YY, et al. Advances of JAZ family in plants[J]. China Ind Econ, 2021, 57(5): 1039-1046.
[32]	Fu J, Wu H, Ma SQ, et al. OsJAZ1 attenuates drought resistance by regulating JA and ABA signaling in rice[J]. Front Plant Sci, 2017, 8: 2108.
[33]	黄文峰, 王立丰, 田维敏. 茉莉酸反应基因转录抑制因子JAZ蛋白家族研究进展[J]. 热带作物学报, 2009, 30(9): 1383-1387.
	Huang WF, Wang LF, Tian WM. Advances in JAZ protein family mediating JA signal pathway[J]. Chin J Trop Crops, 2009, 30(9): 1383-1387.
[34]	闫会转. JAZ2和JAZ7调控茉莉酸介导的转录与代谢重编程的机理研究[D]. 杭州: 浙江大学, 2014.
	Yan HZ. Mechanism of JAZ2 and JAZ7 in regulating jasmonic acid mediated transcription and metabolic reprogramming[D]. Hangzhou: Zhejiang University, 2014.
[35]	安立昆. 玉米茉莉酸信号受体基因家族ZmCOIs功能研究[D]. 南京: 南京农业大学, 2018.
	An LK. Study on the functional of maize jasmonic acid signal receptor gene family ZmCOIs[D]. Nanjing: Nanjing Agricultural University, 2018.
[36]	Saito R, Hayashi K, Nomoto H, et al. Extended JAZ degron sequence for plant hormone binding in jasmonate co receptor of tomato S1COI1 S1JAZ[J]. Sci Rep, 2021, 11(1): 13612. doi: 10.1038/s41598-021-93067-1 pmid: 34193940

引物Primer	引物序列Primer sequence（5'-3'）
HvnJAZ4	AGCCGGCTGGATGGAGAGGGA
HvnJAZ4	TATTATATCGGATCAGATGT
HvnJAZ4 promotor	CATGCTAACTGCTCCTAG
HvnJAZ4 promotor	CCAGCCGGCTCGGGAA
HvnJAZ4 qPCR	TCATCCTGTGACACTAGCCTCC
HvnJAZ4 qPCR	GACTTGCTGTTGTTGTCGTT
HvnJAZ1	ATGGATCTGCTGGA
HvnJAZ1	TTACTGGGCCTT
HvnNINJA1	ATGGAGGATGGCCTTGA
HvnNINJA1	TTAGTTTTGGGCCGAGGC
HvnCOI1b	AGAAAGGGTGGGAGGGAGGAGGA
HvnCOI1b	CCCAAGCGACGAGGGGCAATAAG
18SrRNA	ACGAGTCAGCCTTCGTCGT
18SrRNA	GCGATCTCTGTGCATGATG
HvnJAZ4-GFP	GCTCTAGAAGAAAGGGTGGGAGGGAGGAGGA
HvnJAZ4-GFP	CGGGTACCCTCAGATGTGTAGTTTTGT

引物Primer	引物序列Primer sequence（5'-3'）
HvnJAZ4	AGCCGGCTGGATGGAGAGGGA
HvnJAZ4	TATTATATCGGATCAGATGT
HvnJAZ4 promotor	CATGCTAACTGCTCCTAG
HvnJAZ4 promotor	CCAGCCGGCTCGGGAA
HvnJAZ4 qPCR	TCATCCTGTGACACTAGCCTCC
HvnJAZ4 qPCR	GACTTGCTGTTGTTGTCGTT
HvnJAZ1	ATGGATCTGCTGGA
HvnJAZ1	TTACTGGGCCTT
HvnNINJA1	ATGGAGGATGGCCTTGA
HvnNINJA1	TTAGTTTTGGGCCGAGGC
HvnCOI1b	AGAAAGGGTGGGAGGGAGGAGGA
HvnCOI1b	CCCAAGCGACGAGGGGCAATAAG
18SrRNA	ACGAGTCAGCCTTCGTCGT
18SrRNA	GCGATCTCTGTGCATGATG
HvnJAZ4-GFP	GCTCTAGAAGAAAGGGTGGGAGGGAGGAGGA
HvnJAZ4-GFP	CGGGTACCCTCAGATGTGTAGTTTTGT

元件 Site name	序列 Sequence	功能 Function	数量 Amouny
A-box	CCGTCC	顺式作用调节元件Cis-acting regulatory element	1
ABRE	ACGTG	参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness	5
ARE	AAACCA	对厌氧诱导调节顺式作用元件 Cis-acting regulatory element essential for the anaerobic induction	3
AuxRR-core	GGTCCAT	参与生长素反应的顺式作用调节元件 Cis-acting regulatory element involved in auxin responsiveness	1
Box 4	ATTAAT	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness	1
CAAT-box	CCAAT; CAAAT	启动子和增强子区域中常见的顺式作用元件 Common Cis-acting element in promoter and enhancer regions	10
CAT-box	GCCACT	与分生组织表达相关的顺式作用调控元件 Cis-acting regulatory element related to meristem expression	3
CGTCA-motif	CGTCA	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	2
G-Box	CACGTC	参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness	2
G-box	CACGTC; CACGAC; GCCACGTGGA	参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness	5
GC-motif	CCCCCG	参与缺氧特异性诱导的增强子样元件 Enhancer-like element involved in anoxic specific inducibility	2
I-box	CCATATCCAAT; CGATAAGGCG	光响应元件Part of a light responsive element	2
LTR	CCGAAA	参与低温响应的顺式作用元件 Cis-acting element involved in low-temperature responsiveness	1
MBS	CAACTG	MYB结合位点参与干旱诱导MYB binding site involved in drought-inducibility	1
O2-site	GATGATGTGG	参与醇溶蛋白代谢调节的顺式作用调节元件 Cis-acting regulatory element involved in zein metabolism regulation	1
TATA-box	TATATA; ATATAT; TATA; ACAAAA; TATAA; TATATTTATATTT	启动子核心元件Core promoter element around -30 of transcription start	46
TATC-box	TATCCCA	参与赤霉素反应的顺式作用元件 Cis-acting element involved in gibberellin-responsiveness	1
TC-rich repeats	GTTTTCTTAC	参与防御和压力反应的顺式作用元件 Cis-acting element involved in defense and stress responsiveness	1
TCA-element	CCATCTTTTT; TCAGAAGAGG	参与水杨酸反应性的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness	2
TCCC-motif	TCTCCCT	光响应元件Part of a light responsive element	3
TGACG-motif	TGACG	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	2
Unnamed_5	TGTAATAATATATTTATATT	SEF1因子结合位点SEF1 factor binding site	1

元件 Site name	序列 Sequence	功能 Function	数量 Amouny
A-box	CCGTCC	顺式作用调节元件Cis-acting regulatory element	1
ABRE	ACGTG	参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness	5
ARE	AAACCA	对厌氧诱导调节顺式作用元件 Cis-acting regulatory element essential for the anaerobic induction	3
AuxRR-core	GGTCCAT	参与生长素反应的顺式作用调节元件 Cis-acting regulatory element involved in auxin responsiveness	1
Box 4	ATTAAT	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness	1
CAAT-box	CCAAT; CAAAT	启动子和增强子区域中常见的顺式作用元件 Common Cis-acting element in promoter and enhancer regions	10
CAT-box	GCCACT	与分生组织表达相关的顺式作用调控元件 Cis-acting regulatory element related to meristem expression	3
CGTCA-motif	CGTCA	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	2
G-Box	CACGTC	参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness	2
G-box	CACGTC; CACGAC; GCCACGTGGA	参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness	5
GC-motif	CCCCCG	参与缺氧特异性诱导的增强子样元件 Enhancer-like element involved in anoxic specific inducibility	2
I-box	CCATATCCAAT; CGATAAGGCG	光响应元件Part of a light responsive element	2
LTR	CCGAAA	参与低温响应的顺式作用元件 Cis-acting element involved in low-temperature responsiveness	1
MBS	CAACTG	MYB结合位点参与干旱诱导MYB binding site involved in drought-inducibility	1
O2-site	GATGATGTGG	参与醇溶蛋白代谢调节的顺式作用调节元件 Cis-acting regulatory element involved in zein metabolism regulation	1
TATA-box	TATATA; ATATAT; TATA; ACAAAA; TATAA; TATATTTATATTT	启动子核心元件Core promoter element around -30 of transcription start	46
TATC-box	TATCCCA	参与赤霉素反应的顺式作用元件 Cis-acting element involved in gibberellin-responsiveness	1
TC-rich repeats	GTTTTCTTAC	参与防御和压力反应的顺式作用元件 Cis-acting element involved in defense and stress responsiveness	1
TCA-element	CCATCTTTTT; TCAGAAGAGG	参与水杨酸反应性的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness	2
TCCC-motif	TCTCCCT	光响应元件Part of a light responsive element	3
TGACG-motif	TGACG	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	2
Unnamed_5	TGTAATAATATATTTATATT	SEF1因子结合位点SEF1 factor binding site	1

理化性质Physicochemical property	HvnJAZ4	理化性质Physicochemical property	HvnJAZ4
分子重量Molecular weight/Da	44 055.39	α 螺旋比例Proportions of α helix/%	23.50
总原子数Total number of atoms	6 124	延伸链比例Proportions of extended strand/%	12.47
分子式Formula	C₁₉₀₈H₃₀₃₀N₅₆₈O₆₀₃S₁₂	β-折叠比例Proportions of β turn/%	2.64
亲水系数GRAVY	-0.497	无规则卷曲比例Proportions of random coil/%	61.39
理论等电点Theoretical pI	10.0	亚细胞定位预测Prediction of subcellular localization	细胞核Nucleus
不稳定指数Instability index（II）	71.38	信号肽Signal peptide	无No
跨膜结构Transmembrane structures	无No	磷酸化位点Phosphorylation site	丝氨酸43个
糖基化位点Glycosylation site 脂溶指数Aliphatic index	无No 57.03		苏氨酸11个酪氨酸2个